Antibodies against human IL33R and uses thereof

ABSTRACT

An antibody binding to IL33R characterized in that the heavy chain variable domain comprises a CDR3 region of SEQ ID NO:1, a CDR2 region of SEQ ID NO:2 and a CDR1 region of SEQ ID NO:3 and in that the light chain variable domain comprises a CDR3 region of SEQ ID NO:4, a CDR2 region of SEQ ID NO:5 and a CDR1 region of SEQ ID NO:6 or a chimeric, humanized or T cell epitope depleted antibody variant thereof has advantageous properties for the treatment of inflammatory diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims of priority under 35 USC §119(a) toEuropean patent application number EP11155684.1, filed 23 Feb. 2011, thecontents of which is incorporated herein by reference.

SEQUENCE LISTING

A sequence listing comprising SEQ ID NOS: 1-34 is attached hereto. Eachsequence provided in the sequence listing is incorporated herein byreference, in its entirety, for all purposes.

TECHNICAL FIELD

The present invention relates to antibodies against human IL33R (IL33Rantibody), methods for their production, pharmaceutical compositionscontaining said antibodies, and uses thereof.

BACKGROUND OF THE INVENTION

Human IL33 is an interleukin-1-like cytokine of the IL-1 family thatsignals via the IL-1 receptor-related IL33 receptor (synonyms of thereceptor: IL1RL1, T1/ST2) and induces T helper type 2-associatedcytokines. Synonyms of IL33 (Swiss-Prot Acc. No. O95760) areinterleukin-1 family member 11 (IL-1F11) and nuclear factor from highendothelial venules (NF-HEV). NF-HEV is described in Baekkevold, E. S.,et al., Am. J. Pathol. 163 (2003) 69-79. IL33 is described by Schmitz,J., et al., Immunity 23 (2005) 479-490.

Human IL33 Receptor, IL33R, (synonym for ILRL1; SwissProt Acc No.Q01638, other names are ST2, T1/ST2, Fit-1 and DER4) is induced bygrowth stimulation in fibroblastic cells, and can also be induced byantigen stimulation in Th2 cells. According to the invention IL33R andST2 denotes human IL33R. Tominaga, S., et al., (FEBS Lett. 258 (1989)301-304; Biochim. Biophys. Acta. 1171 (1992) 215-218) and Yanagisawa,K., (FEBS Lett. 318 (1993) 83-87) identified human ST2 (the secretedform), ST2L (the transmembrane receptor form) and ST2V (variantGlu-78,). Human ST2 is only expressed in growth-stimulated BALB/c-3T3cells and a member of the primary response gene family induced by growthfactors. ST2 encodes a protein similar in sequence to the extracellularportion of human interleukin 1 receptor, both types 1 and 2. Studieswith IL33R knockout mice suggest that IL33R is involved in early eventsof Th2-response (Kropf, P., et al., Infect. Immunity 70 (2002)5512-5520; Hoshino, K., et al., J. Exp. Med. 190 (1999) 1541-1548; Senn,et al., Eur. J. Immunol. 30 (2000) 1929-1938; Townsend, M. J., et al.,J. Exp. Med. 191 (2000) 1069-1076). ST2 is assumed to be a marker,activator and regulator of Th2 immunity (Kumar, R. K., et al., Clin.Exp. Allergy 32 (2002) 1394-1396).

Anti-IL33R antibodies and their role in immune function were describedin a number of publications. Anti-human ST2 antibody Mab523 andpolyclonal antibody AF523 are commercially availably from R&D Systems(which can be found on the world wide web with a URL address ofrndsystems.com). Anti-human ST2 antibody HB12 is commercially availablyfrom antibodies-online GmbH, Germany and MBL Int. Corp. (which can befound on the world wide web with a URL address of mblintl.com).Anti-IL33R antibodies resulted in decreased Th2-type immune responses.The antibody inhibited eosinophil infiltration, IL-5 production, andIgE-production. The evaluation of the role of ST2 in animal models forasthma resulted in increased expression of murine IL33R on CD4+ T cells,indicating a role for IL33R in allergic or asthmatic responses (Löhning,M., et al., Proc. Natl. Acad. Sci. USA 95 (1998) 6930-6935 and Xu, D.,et al., J. Exp. Med. 187 (1998) 787-794; Coyle, A. J., et al., J. Exp.Med. 190 (1999) 895-902). Meisel, C., et al., J. Immunol. 166 (2001)3143-3150 investigate the regulation and function of T1/ST2 expressionon CD4⁺ T cells and the induction of type 2 cytokine production byT1/ST2 cross-linking. Löhning, M., et al., generate an anti-mouse ST2antibody in rats. Pretreatment with 20 μg (approx 0.0.8 mg/kg) of suchan antibody 1 hr before allergen provocation reduced the number ofeosinophils in the mouse airway by 70%. Kumar, S., et al., (Biochem.Biophys. Res. Comm. 235 (1997) 474-478 and J. Biol. Chem. 270 (1995)27905-27913) describe the expression of ST2 protein detected byimmunoprecipitation using a rabbit polyclonal antibody generated againstpurified soluble ST2 receptor expressed in Drosophila. Studies withBALB/c mice revealed that treating with anti-IL33 antibody inducedhigher Th1-type response. An ELISA system to quantify human ST2 proteinin sera of patients was described by Kuroiwa, K., et al., Hybridoma 19(2000) 151-159. Anti-IL33R antibodies also reduce effects due toinfections with RSV (Walzl, et al., J. Exp. Med. 193 (2001) 785-792).Anti-IL33R antibodies were also investigated in an animal model ofarthritis (Leung, B. P., et al., J. Immunol. 173 (2004) 145-150; Walzl,et al., J. Exp. Med. 193 (2001) 785-792). Smithgall, M. D., et al., Int.Immunol. 20 (2008) 1019-1030 investigated interferon γ levels in NKcells in the presence of an anti-huST2 antibody. IL33R and/or antibodiesagainst IL33R are mentioned in WO 2005/079844, U.S. Pat. No. 7,087,396,WO 2001/021641, WO 2002/038794, WO 2003/094856. Oboki, K., et al.,Allergology Int. 59 (2010) 143-160, review the role of IL33 and IL33receptors in host defense and diseases and the effects of anti-ST2antibody, soluble ST2 and anti-IL33 antibody on mouse airwayinflammation.

SUMMARY OF THE INVENTION

The invention comprises an antibody binding to IL33R, characterized inthat the heavy chain variable domain comprises a CDR3 region of SEQ IDNO:1, a CDR2 region of SEQ ID NO:2 and a CDR1 region of SEQ ID NO:3 andin that the light chain variable domain comprises a CDR3 region of SEQID NO:4, a CDR2 region of SEQ ID NO:5 and a CDR1 region of SEQ ID NO:6.Preferably the antibody is characterized in that the heavy chainvariable domain comprises SEQ ID NO:7. Preferably the antibody ischaracterized in that the heavy chain variable domain comprises SEQ IDNO:7 and the light chain variable domain comprises SEQ ID NO:8.Preferably the antibody binding to IL33R and being characterized by theabove mentioned amino acid sequences and amino acid sequence fragmentsis of human IgG1 isotype modified in the hinge region at amino acidposition 216-240, preferably at amino acid position 220-240, betweenC_(H)1 and C_(H)2 and/or in the second inter-domain region at amino acidposition 327-331 between C_(H)2 and C_(H)3. Preferably the antibodycomprises mutations L234A (alanine instead of leucine at amino acidposition 234) and L235A. A preferred heavy chain constant regionincluding mutations L234A and L235A is shown in SEQ ID NO:9. Preferablythe antibody binding to IL33R and being characterized by the abovementioned amino acid sequences and amino acid sequence fragments is ofhuman IgG4 isotype modified in the hinge region at amino acid position216-240, preferably at amino acid position 220-240, between C_(H)1 andC_(H)2 and/or in the second inter-domain region at amino acid position327-331 between C_(H)2 and C_(H)3. Preferably the antibody comprisesmutations L235E (glutamic acid instead of leucine at amino acid position235) and S228P (proline instead of serine at amino acid position 228).

Antibody ra170 (Mab ra170) is a preferred embodiment of the invention. Afurther embodiment of the invention is a chimeric, humanized or T cellepitope depleted antibody variant of antibody ra170.

Preferred humanized antibody variants of antibody ra170 arecharacterized in that the heavy chain variable domain comprises a CDR3region of SEQ ID NO: 24, a CDR2 region of SEQ ID NO:23 and a CDR1 regionof SEQ ID NO:22 and in that the light chain variable domain comprises aCDR3 region of SEQ ID NO: 33, a CDR2 region of SEQ ID NO:32 and a CDR1region of SEQ ID NO:31, or are characterized in that the heavy chainvariable domain comprises a CDR3 region of SEQ ID NO: 28, a CDR2 regionof SEQ ID NO:27 and a CDR1 region of SEQ ID NO:26 and in that the lightchain variable domain comprises a CDR3 region of SEQ ID NO: 33, a CDR2region of SEQ ID NO:32 and a CDR1 region of SEQ ID NO:31. Preferably thehumanized antibody is characterized in that the heavy chain variabledomain comprises SEQ ID NO:21 or 25. Preferably the humanized antibodyis characterized in that the light chain variable domain comprises SEQID NO:30.

The antibody binds specifically to IL33R with an affinity of 10⁻¹⁰M orlower.

The invention relates further to an antibody binding to IL33R and beingcharacterized by binding to the same IL33R epitope to which monoclonalantibody ra170 binds. The antibody binds to IL33R with an affinity of atleast 10⁻⁸ M⁻¹ to 10⁻¹² M⁻¹, is preferably of human IgG1 isotypemodified in the hinge region at amino acid position 216-240, preferablyat amino acid position 220-240, between C_(H)1 and C_(H)2 and/or in thesecond inter-domain region at amino acid position 327-331 between C_(H)2and C_(H)3. Preferably the antibody is of human IgG1 isotype comprisingmutations L234A and L235A or of human IgG4 isotype comprising mutationsL235E and S228P.

Preferably the antibody is a humanized or human antibody. Preferably theantibody according to the invention inhibits binding of IL33 to IL33Rwith an IC50 value of 0.32 nM for human IL33/IL33R and 0.13 nM forcynomolgus IL33/IL33R.

Antibodies according to the invention preferably show IC₅₀ values of 5nM or lower in the eosoinophil assay, mast cell assay, Th2 assay,basophil assay (IL-5), Such antibodies are especially useful in thetreatment of rheumatoid arthritis, asthma or ulcerative colitis.

A further embodiment of the invention is a pharmaceutical compositioncomprising an antibody according to the invention. Preferably thepharmaceutical composition comprises an antibody characterized bybinding to the same IL33R epitope to which monoclonal antibody ra170binds. Preferably the antibody of the pharmaceutical composition bindsto IL33R with an affinity of at least 10⁻⁸ M⁻¹ to 10⁻¹² M⁻¹, ispreferably of human IgG1 isotype modified in the hinge region at aminoacid position 216-240, preferably at amino acid position 220-240,between C_(H)1 and C_(H)2 and/or in the second inter-domain region atamino acid position 327-331 between C_(H)2 and C_(H)3. Preferably theantibody is of human IgG1 isotype comprising mutations L234A (alanineinstead of leucine at amino acid position 234) and L235A or of humanIgG4 isotype comprising mutations L235E and S228P.

A further embodiment of the invention is the use of an antibodyaccording to the invention for the manufacture of a pharmaceuticalcomposition. Preferably the pharmaceutical composition comprises anantibody characterized by binding to the same IL33R epitope to whichmonoclonal antibody ra170 binds. Preferably the antibody of thepharmaceutical composition binds to IL33R with an affinity of at least10⁻⁸ M⁻¹ to 10⁻¹² M⁻¹, is preferably of human IgG1 isotype modified inthe hinge region at amino acid position 216-240, preferably at aminoacid position 220-240, between C_(H)1 and C_(H)2 and/or in the secondinter-domain region at amino acid position 327-331 between C_(H)2 andC_(H)3. Preferably the antibody is of human IgG1 isotype comprisingmutations L234A (alanine instead of leucine at amino acid position 234)and L235A or is of human IgG4 isotype comprising mutations L235E andS228P.

A further embodiment of the invention is the use of an antibodyaccording to the invention for the treatment of ulcerative colitis orasthma. A further embodiment of the invention is a method for themanufacture of a pharmaceutical composition comprising an antibodyaccording to the invention. Preferably the pharmaceutical compositioncomprises an antibody characterized by binding to the same IL33R epitopeto which monoclonal antibody ra170 binds. Preferably the antibody of thepharmaceutical composition binds to IL33R with an affinity of at least10⁻⁸ M⁻¹ to 10⁻¹² M⁻¹, is preferably of human IgG1 isotype modified inthe hinge region at amino acid position 216-240, preferably at aminoacid position 220-240, between C_(H)1 and C_(H)2 and/or in the secondinter-domain region at amino acid position 327-331 between C_(H)2 andC_(H)3. Preferably the antibody is of human IgG1 isotype comprisingmutations L234A (alanine instead of leucine at amino acid position 234)and L235A or is of human IgG4 isotype comprising mutations L235E andS228P.

A further embodiment of the invention is a nucleic acid encoding a heavychain of an antibody binding to IL33R, characterized in comprising aheavy chain CDR3 region of SEQ ID NO:1 and preferably mutations L234Aand L235A in the IgG1 heavy chain constant domain. Preferably theantibody comprises in addition a heavy chain CDR2 region of SEQ ID NO:2and a CDR1 region of SEQ ID NO:3. A further embodiment of the inventionis a nucleic acid encoding a light chain of an antibody binding toIL33R, characterized by comprising a light chain CDR3 region of SEQ IDNO:4 and preferably mutations L234A and L235A in the IgG1 heavy chainconstant domain. Preferably the antibody comprises in addition a lightchain CDR2 region of SEQ ID NO:5 and a CDR1 region of SEQ ID NO:6. Afurther embodiment of the invention is a nucleic acid encoding anantibody according to the invention characterized by comprising a heavychain variable domain of SEQ ID NO: 7 and a variable light chain domainof SEQ ID NO:8 and preferably mutations L234A and L235A in the heavychain human IgG1 constant domain or mutations L235E and S228P in theheavy chain human IgG4 constant domain.

A further embodiment of the invention is a nucleic acid encoding a heavychain of an antibody binding to IL33R, characterized in comprising aheavy chain CDR3 region of SEQ ID NO:24 and preferably mutations L234Aand L235A in the IgG1 heavy chain constant domain. Preferably theantibody comprises in addition a heavy chain CDR2 region of SEQ ID NO:23and a CDR1 region of SEQ ID NO:22. A further embodiment of the inventionis a nucleic acid encoding a light chain of an antibody binding toIL33R, characterized by comprising a light chain CDR3 region of SEQ IDNO:33 and preferably mutations L234A and L235A in the IgG1 heavy chainconstant domain. Preferably the antibody comprises in addition a lightchain CDR2 region of SEQ ID NO:2 and a CDR1 region of SEQ ID NO:31. Afurther embodiment of the invention is a nucleic acid encoding anantibody according to the invention characterized by comprising a heavychain variable domain of SEQ ID NO: 21 and a variable light chain domainof SEQ ID NO:30 and preferably mutations L234A and L235A in the heavychain human IgG1 constant domain or mutations L235E and S228P in theheavy chain human IgG4 constant domain.

A further embodiment of the invention is a nucleic acid encoding a heavychain of an antibody binding to IL33R, characterized in comprising aheavy chain CDR3 region of SEQ ID NO:28 and preferably mutations L234Aand L235A in the IgG1 heavy chain constant domain. Preferably theantibody comprises in addition a heavy chain CDR2 region of SEQ ID NO:27and a CDR1 region of SEQ ID NO:26. A further embodiment of the inventionis a nucleic acid encoding a light chain of an antibody binding toIL33R, characterized by comprising a light chain CDR3 region of SEQ IDNO:33 and preferably mutations L234A and L235A in the IgG1 heavy chainconstant domain. Preferably the antibody comprises in addition a lightchain CDR2 region of SEQ ID NO:2 and a CDR1 region of SEQ ID NO:31. Afurther embodiment of the invention is a nucleic acid encoding anantibody according to the invention characterized by comprising a heavychain variable domain of SEQ ID NO: 25 and a variable light chain domainof SEQ ID NO:30 and preferably mutations L234A and L235A in the heavychain human IgG1 constant domain or mutations L235E and S228P in theheavy chain human IgG4 constant domain.

The antibody according to the invention is preferably characterized inthat the constant chains are of human origin. Such constant chains arewell known in the state of the art and e.g., described by Kabat (Kabat,E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed.,Public Health Service, National Institutes of Health, Bethesda, Md.(1991), and Johnson, G. and Wu, T. T., Nucleic Acids Res. 28 (2000)214-218). For example, a useful human heavy chain constant regioncomprises an amino acid sequence of SEQ ID NO: 9 (human IgG1 includingthe mutations L234A and L235A) or of SEQ ID NO: 29 (human IgG4 includingthe mutations L235E and S228P). For example, a useful human light chainconstant region comprises an amino acid sequence of a kappa-light chainconstant region of SEQ ID NO: 12 or 34. It is further preferred that theantibody is of mouse origin and comprises the antibody variable sequenceframe of a mouse antibody according to Kabat (Kabat, E. A., et al.,Sequences of Proteins of Immunological Interest, 5th ed., Public HealthService, National Institutes of Health, Bethesda, Md. (1991); andJohnson, G. and Wu, T. T., Nucleic Acids Res. 28 (2000) 214-218).

The antibody according to the invention is especially characterized byinhibiting the binding of IL33 to IL33R and therefore of inhibitingsignaling via IL33R/IL-1RacP signaling complex.

The antibody according to the invention is preferably of human isotypeIgG1. Preferred γ1 heavy chain constant regions are shown in SEQ IDNO:10 or 29 and in SEQ ID NO:11 without L234A and L235A mutations. Apreferred K light chain constant region is shown in SEQ ID NO:12 or 34.

The antibody according to the invention is preferably characterized bynot binding human complement factor C1q and avoid therefore CDC effectorfunction.

The antibody according to the invention is preferably of human IgG1isotype modified in the hinge region at amino acid position 216-240,preferably at amino acid position 220-240, between C_(H)1 and C_(H)2and/or in the second inter-domain region at amino acid position 327-331between C_(H)2 and C_(H)3. The antibody according to the invention ispreferably characterized by being of human IgG1 isotype, containing atleast one mutation in, L234 (leucine at amino acid position 234), L235,D270, N297, E318, K320, K322, P331, and/or P329 (numbering according toEU index). Preferably the antibody is of human IgG1 isotype comprisingmutations L234A (alanine instead of leucine at amino acid position 234)and L235A or is of human IgG4 isotype comprising mutations L235E andS228P.

The invention further provides expression vectors containing nucleicacid according to the invention capable of expressing said nucleic acidin a prokaryotic or eukaryotic host cell, and host cells containing suchvectors for the recombinant production of such an antibody. Theinvention further comprises a prokaryotic or eukaryotic host cellcomprising a vector according to the invention. The invention furthercomprises a method for the production of a recombinant human orhumanized antibody according to the invention, characterized byexpressing a nucleic acid according to the invention in a prokaryotic oreukaryotic host cell and recovering said antibody from said cell or thecell culture supernatant. The invention further comprises the antibodyobtainable by such a recombinant method.

Antibodies according to the invention show benefits for patients in needof an IL33R targeting therapy. The antibodies according to the inventionhave new and inventive properties causing especially a benefit for apatient suffering from such an immunological disease, especiallysuffering from rheumatoid arthritis, ulcerative colitis or asthma. Theantibodies according to the invention are not causing susceptibility forstaphylococcal and enteric bacterial infections of the treated patient.The invention further provides a method for treating a patient sufferingfrom rheumatoid arthritis, ulcerative colitis or asthma comprisingadministering to a patient diagnosed as having such a disease (andtherefore being in need of such a therapy) an effective amount of anantibody binding to IL33R according to the invention. The antibody isadministered preferably in a pharmaceutical composition. A furtherembodiment of the invention is a method for the treatment of a patientsuffering from rheumatoid arthritis, ulcerative colitis or asthma,characterized by administering to the patient an antibody according tothe invention. The invention further comprises the use of an antibodyaccording to the invention for the treatment of a patient suffering fromrheumatoid arthritis, ulcerative colitis or asthma and for themanufacture of a pharmaceutical composition according to the invention.In addition, the invention comprises a method for the manufacture of apharmaceutical composition according to the invention.

The invention further comprises a pharmaceutical composition comprisingan antibody according to the invention, optionally together with abuffer and/or an adjuvant useful for the formulation of antibodies forpharmaceutical purposes. The invention further provides pharmaceuticalcompositions comprising an antibody according to the invention in apharmaceutically acceptable carrier. In one embodiment, thepharmaceutical composition may be included in an article of manufactureor kit.

DETAILED DESCRIPTION OF THE INVENTION

The term “antibody” encompasses the various forms of antibody structuresincluding but not being limited to whole antibodies and antibodyfragments. The antibody according to the invention is preferably ahumanized antibody, chimeric antibody, or further genetically engineeredantibody as long as the characteristic properties according to theinvention are retained. “Antibody fragments” comprise a portion of afull length antibody, preferably the variable domain thereof, or atleast the antigen binding site thereof. Examples of antibody fragmentsinclude diabodies, single-chain antibody molecules, and multispecificantibodies formed from antibody fragments. scFv antibodies are, e.g.,described in Huston, J. S., Methods in Enzymol. 203 (1991) 46-88. Inaddition, antibody fragments comprise single chain polypeptides havingthe characteristics of a V_(H) domain, namely being able to assembletogether with a V_(L) domain, or of a V_(L) domain binding to IL33R,namely being able to assemble together with a V_(H) domain to afunctional antigen binding site and thereby providing the properties ofan antibody according to the invention. The terms “monoclonal antibody”or “monoclonal antibody composition” as used herein refer to apreparation of antibody molecules of a single amino acid composition.The term “humanized antibody” refers to antibodies in which theframework and/or “complementary determining regions” (CDR) have beenmodified to comprise the CDR of an immunoglobulin of different speciesas compared to that of the parent immunoglobulin. In a preferredembodiment, a mouse CDR is grafted into the framework region of a humanantibody to prepare the “humanized antibody”. See, e.g., Riechmann, L.,et al., Nature 332 (1988) 323-327; and Neuberger, M. S., et al., Nature314 (1985) 268-270.

The term “binding to IL33R” as used herein means binding of the antibodyto immobilized human IL33R in an ELISA binding assay. Binding is foundif the antibody causes a signal greater than average +3 standarddeviations or more of the control without antibody at an antibodyconcentration of greater than 12 ng/ml.

The term “affinity” refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Specificillustrative and exemplary embodiments for measuring binding affinityare described in the following.

The term “epitope” denotes a protein determinant capable of specificallybinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually epitopes have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and nonconformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents. Preferably an antibody according to the inventionbinds specifically to native but not to denatured IL33R. The IL33Rantibody of the invention binds to the same epitope on IL33R to whichantibody Mab ra170 binds. The epitope binding property of an IL33Rantibody of the present invention may be determined using techniquesknown in the art. The IL33R antibody is tested by an in vitrocrossblocking binding assay to determine the ability of the testantibody to hinder the binding of antibody Mab ra170 to IL33R. If thereis a displacement of the test antibody by antibody Mab ra170 for atleast 15%, then the epitopes are in near proximity.

The “variable domain” (variable domain of a light chain (V_(L)),variable domain of a heavy chain (V_(H))) as used herein denotes each ofthe pair of light and heavy chain domains which are involved directly inbinding of the antibody to the antigen. The variable light and heavychain domains have the same general structure and each domain comprisesfour framework (FR) regions whose sequences are widely conserved,connected by three “hypervariable regions” (or complementary determiningregions, CDRs). The framework regions adopt a (β-sheet conformation andthe CDRs may form loops connecting the β-sheet structure. The CDRs ineach chain are held in their three-dimensional structure by theframework regions and form together with the CDRs from the other chainthe antigen binding site. The antibody's heavy and light chain CDR3regions play a particularly important role in the bindingspecificity/affinity of the antibodies according to the invention andtherefore provide a further object of the invention.

The term “antigen-binding portion of an antibody” when used hereinrefers to the amino acid residues of an antibody which are responsiblefor antigen-binding. The antigen-binding portion of an antibodycomprises amino acid residues from the “complementary determiningregions” or “CDRs”. “Framework” or “FR” regions are those variabledomain regions other than the hypervariable region residues as hereindefined. Therefore, the light and heavy chain variable domains of anantibody comprise from N- to C-terminus the domains FR1, CDR1, FR2,CDR2, FR3, CDR3, and FR4. Especially, CDR3 of the heavy chain is theregion which contributes most to antigen binding and defines theantibody's properties. CDR and FR regions are determined according tothe standard definition of Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) and/or those residues from a“hypervariable loop”.

The term “amino acid” as used within this application denotes the groupof naturally occurring carboxy-amino acids comprising alanine (threeletter code: ala, one letter code: A), arginine (arg, R), asparagine(asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q),glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine(ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M),phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine(thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

The terms “nucleic acid” or “nucleic acid molecule”, as used herein, areintended to include DNA molecules and RNA molecules. A nucleic acidmolecule may be single-stranded or double-stranded, but preferably isdouble-stranded DNA. A nucleic acid is “operably linked” when it isplaced into a functional relationship with another nucleic acid. Forexample, DNA for a presequence or secretory leader is operably linked toDNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Generally, “operable linked” means that the DNA sequences being linkedare colinear, and, in the case of a secretory leader, contiguous and inreading frame. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, synthetic oligonucleotide adaptors or linkers areused in accordance with conventional practice. As used herein, theexpressions “cell”, “cell line”, and “cell culture” are usedinterchangeably and all such designations include progeny. Thus, thewords “transformants” and “transformed cells” include the primarysubject cell and cultures derived therefrom without regard for thenumber of transfers. It is also understood that all progeny may not beprecisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded.

The “Fc part” of an antibody is not involved directly in binding of anantibody to an antigen, but exhibits various effector functions. An “Fcpart of an antibody” is a term well known to the skilled artisan anddefined on the basis of papain cleavage of antibodies. Depending on theamino acid sequence of the constant region of their heavy chains,antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. Accordingto the heavy chain constant regions the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ respectively. The Fc partof an antibody is directly involved in ADCC (antibody-dependentcell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity)based on complement activation, C1q binding and Fc receptor binding.Complement activation (CDC) is initiated by binding of complement factorC1q to the Fc part of most IgG antibody subclasses. While the influenceof an antibody on the complement system is dependent on certainconditions, binding to C1q is caused by defined binding sites in the Fcpart. Such binding sites are known in the state of the art and describedby, e.g., Boackle, R. J., et al., Nature 282 (1979) 742-743; Lukas, T.J., et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R. and Cebra,J. J., Mol. Immunol. 16 (1979) 907-917; Burton, D. R., et al., Nature288 (1980) 338-344; Thommesen, J. E., et al., Mol. Immunol. 37 (2000)995-1004; Idusogie, E. E., et al., J. Immunol. 164 (2000) 4178-4184;Hezareh, M., et al., J. Virology 75 (2001) 12161-12168; Morgan, A., etal., Immunology 86 (1995) 319-324; EP 0 307 434. Such binding sites are,e.g., L234, L235, D270, N297, E318, K320, K322, P331, and P329(numbering according to EU index of Kabat, Kabat, E. A., et al.,Sequences of Proteins of Immunological Interest, 5th ed., Public HealthService, National Institutes of Health, Bethesda, Md. (1991)).Antibodies of subclass IgG1, IgG2 and IgG3 usually show complementactivation and C1q binding, whereas IgG4 does not activate thecomplement system and does not bind C1q.

The antibody according to the invention comprises an Fc part from humanorigin which is Fc part of a human antibody of the subclass IgG1 orIgG4. For the Fc part of an antibody according to the inventionpreferably no C1q binding as defined below can be detected.

The invention therefore comprises an antibody according to theinvention, characterized in that said antibody binds IL33R, contains anFc part from human origin, and does not bind human complement factor C1qand therefore avoids CDC effector function.

Preferably an antibody according to the invention is in regard to Fcγreceptor binding of human IgG1 or IgG2 subclass, with a mutation inL234, L235, and/or D265, and/or contains the PVA236 mutation. Preferredare the mutations L234A, L235A, L235E, and/or PVA236 (PVA236 means thatthe amino acid sequence ELLG (given in one letter amino acid code) fromamino acid position 233 to 236 of IgG1 or EFLG of IgG4 is replaced byPVA). The present invention thus provides an antibody according to theinvention being characterized in that said antibody is an antibody ofhuman subclass IgG1, containing at least one mutation in L234, L235,D270, N297, E318, K320, K322, P331 and/or P329. In one embodiment theantibody is a human antibody. In another embodiment the antibody is ahumanized antibody. In one embodiment the present invention provides anantibody according to the invention, containing an Fc part derived fromhuman origin, and being characterized in that said antibody is anantibody of human subclass IgG1, containing at least one mutation inL234, L235, D270, N297, E318, K320, K322, P331 and wherein the antibodybinds to IL33R with a K_(D) value of less than 10⁻⁸ M in a BIAcoreassay. In another embodiment the K_(D) range is 10⁻¹¹ to 10⁻⁹ M.

C1q binding can be measured according to Idusogie, E. E., et al., J.Immunol. 164 (2000) 4178-4184. No C1q binding according to the inventionis characterized in that if in such an assay wherein an ELISA plate iscoated with different concentrations of the antibody, human C1q isadded. C1q binding is detected by an antibody directed against human C1qfollowed by peroxidase-labeled conjugate detection with peroxidasesubstrate ABTS® (2,2′-Azino-di-[3-ethylbenzthiazolinesulfonate]). No C1qbinding according to the invention is found if the optical density (OD)at 405 nm is for the test antibody lower than 0.05 at an antibodyconcentration of 10 μg/ml.

The antibody according to the invention is preferably characterized inthat the constant chains are of human origin. Such constant chains arewell known in the state of the art and described, e.g., by Kabat (Kabat,E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed.,Public Health Service, National Institutes of Health, Bethesda, Md.(1991); and Johnson, G., and Wu, T. T., Nucleic Acids Res. 28 (2000)214-218). For example, a useful human heavy chain constant regioncomprises SEQ ID NO: 10, 11 or 29. For example, a useful human lightchain constant region comprises an amino acid sequence of a kappa-lightchain constant region of SEQ ID NO: 12 or 34.

A further embodiment of the invention is a nucleic acid encoding a heavyand a light chain of an antibody according to the invention.

The invention comprises a method for the treatment of a patient in needof therapy, characterized by administering to the patient atherapeutically effective amount of an antibody according to theinvention. The invention comprises the use of an antibody according tothe invention for therapy. The invention comprises the use of anantibody according to the invention for the preparation of a medicamentfor the prophylaxis and treatment especially of inflammatory disorders.The invention comprises the use of an antibody according to theinvention for the treatment of inflammatory diseases, preferably for thetreatment of rheumatoid arthritis, ulcerative colitis and asthma.

The antibodies according to the invention include, in addition, suchantibodies having “conservative sequence modifications” (variantantibodies), nucleotide and amino acid sequence modifications which donot affect or alter the above-mentioned characteristics of the antibodyaccording to the invention. Modifications can be introduced by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis. Conservative amino acid substitutions includeones in which the amino acid residue is replaced with an amino acidresidue having a similar side chain. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine), beta-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine). Thus, a predicted nonessential amino acidresidue in a human anti-IL33R antibody can be preferably replaced withanother amino acid residue from the same side chain family. A “variant”anti-IL33R antibody, refers therefore herein to a molecule which differsin amino acid sequence from a “parent” anti-IL33R antibody amino acidsequence by up to ten, preferably from about two to about five,additions, deletions and/or substitutions in one or more variable regionof the parent antibody. Amino acid substitutions can be performed bymutagenesis based upon molecular modeling as described by Riechmann, L.,et al., Nature 332 (1988) 323-327 and Queen, C., et al., Proc. Natl.Acad. Sci. USA 86 (1989) 10029-10033.

A further embodiment of the invention is a method for the production ofan antibody against IL33R which does not bind Fcγ receptor and/or C1q,characterized in that the sequence of a nucleic acid encoding the heavychain of a human IgG1 type antibody binding to IL33R is modified in sucha manner that said modified antibody does not bind C1q and/or Fcγreceptor, said modified nucleic acid and the nucleic acid encoding thelight chain of said antibody are inserted into an expression vector,said vector is inserted in a eukaryotic host cell, the encoded proteinis expressed and recovered from the host cell or the supernatant.

Identity or homology with respect to the sequence is defined herein asthe percentage of amino acid residues in the candidate sequence that areidentical with the parent sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity. None of N-terminal, C-terminal, or internal extensions,deletions, or insertions into the antibody sequence shall be construedas affecting sequence identity or homology. The variant retains theability to bind human IL33R and preferably has properties, which aresuperior to those of the parent antibody. For example, the variant mayhave reduced side effects during treatment.

The “parent” antibody comprises the CDR regions of antibody ra170 and ispreferably used for the preparation of the variant. Preferably, theparent antibody has a human framework region and, if present, has ahuman antibody constant region or human antibody constant domains. Forexample, the parent antibody may be a humanized or a human antibody.

The antibodies according to the invention are preferably produced byrecombinant means. Such methods are widely known in the state of the artand comprise protein expression in prokaryotic and eukaryotic cells withsubsequent isolation of the antibody polypeptide and usuallypurification to a pharmaceutically acceptable purity. For the proteinexpression nucleic acids encoding light and heavy chains or fragmentsthereof are inserted into expression vectors by standard methods.Expression is performed in appropriate prokaryotic or eukaryotic hostcells, such as CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COScells, yeast, or E. coli cells, and the antibody is recovered from thecells (from the supernatant or after cells lysis). Recombinantproduction of antibodies is well-known in the state of the art anddescribed, for example, in the review articles of Makrides, S. C.,Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., ProteinExpr. Purif. 8 (1996) 271-282; Kaufman, R. J., Mol. Biotechnol. 16(2000) 151-160; Werner, R. G., Drug Res. 48 (1998) 870-880. Theantibodies may be present in whole cells, in a cell lysate, or in apartially purified, or substantially pure form. Purification isperformed in order to eliminate other cellular components or othercontaminants, e.g., other cellular nucleic acids or proteins, bystandard techniques, including column chromatography and others wellknown in the art. See Ausubel, F., et al., ed., Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York(1987). Expression in NS0 cells is described by, e.g., Barnes, L. M., etal., Cytotechnology 32 (2000) 109-123; Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning ofvariable domains is described by Orlandi, R., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; Norderhaug, L., et al., J. Immunol. Methods 204(1997) 77-87. A preferred transient expression system (HEK 293) isdescribed by Schlaeger, E.-J. and Christensen, K., in Cytotechnology 30(1999) 71-83, and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)191-199. Monoclonal antibodies are suitably separated from the culturemedium by conventional immunoglobulin purification procedures such as,for example, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. DNA and RNAencoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures. The hybridoma cells can serve as a sourceof such DNA and RNA. Once isolated, the DNA may be inserted intoexpression vectors, which are then transfected into host cells, such asHEK 293 cells, CHO cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of recombinantmonoclonal antibodies in the host cells.

Amino acid sequence variants of human IL33R antibody are prepared byintroducing appropriate nucleotide changes into the antibody encodingDNA, or by peptide synthesis. Such modifications can be performed,however, only in a very limited range, e.g., as described above. Forexample, the modifications do not alter the above-mentioned antibodycharacteristics such as the IgG isotype and epitope binding, but mayimprove the yield of the recombinant production, protein stability, orfacilitate the purification. Any cysteine residue not involved inmaintaining the proper conformation of the anti-IL33R antibody may alsobe substituted, generally with serine, to improve the oxidativestability of the molecule and to prevent aberrant crosslinking.Conversely, cysteine bond(s) may be added to the antibody to improve itsstability (particularly where the antibody is an antibody fragment suchas an Fv fragment). Another type of amino acid variant of the antibodyalters the original glycosylation pattern of the antibody. By “altering”is meant removing one or more carbohydrate moieties found in theantibody and/or adding one or more glycosylation sites that are notpresent in the antibody. Glycosylation of antibodies is typicallyN-linked. N-linked refers to the attachment of the carbohydrate moietyto the side chain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. Addition ofglycosylation sites to the antibody is conveniently accomplished byaltering the amino acid sequence such that it contains one or more ofthe above-described tripeptide sequences (for N-linked glycosylationsites).

Nucleic acid molecules encoding amino acid sequence variants ofanti-IL33R antibody are prepared by a variety of methods known in theart. These methods include, but are not limited to, isolation from anatural source (in the case of naturally occurring amino acid sequencevariants) or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of humanized anti-IL33Rantibody.

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192; 4,179,337.

The heavy and light chain variable domains according to the inventionare combined with sequences of promoter, translation initiation,constant region, 3′ untranslated region, polyadenylation, andtranscription termination to form expression vector constructs. Theheavy and light chain expression constructs can be combined into asingle vector, co-transfected, serially transfected, or separatelytransfected into host cells which are then fused to form a single hostcell expressing both chains.

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination ofmonoclonal antibodies, or the antigen-binding portion thereof, of thepresent invention, formulated together with a pharmaceuticallyacceptable carrier. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption/resorptiondelaying agents, and the like that are physiologically compatible.Preferably, the carrier is suitable for injection or infusion. Acomposition of the present invention can be administered by a variety ofmethods known in the art. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. Pharmaceutically acceptable carriers include sterileaqueous solutions or dispersions and sterile powders for the preparationof sterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art. Inaddition to water, the carrier can be, for example, an isotonic bufferedsaline solution. Regardless of the route of administration selected, thecompounds of the present invention, which may be used in a suitablehydrated form, and/or the pharmaceutical compositions of the presentinvention, are formulated into pharmaceutically acceptable dosage formsby conventional methods known to those of skill in the art. Actualdosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient (effectiveamount). The selected dosage level will depend upon a variety ofpharmacokinetic factors including the activity of the particularcompositions of the present invention employed, or the ester, salt oramide thereof, the route of administration, the time of administration,the rate of excretion of the particular compound being employed, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

The invention comprises the use of the antibodies according to theinvention for the treatment of a patient suffering from rheumatoidarthritis, ulcerative colitis or asthma.

A further embodiment of the invention is the use of an anti-IL33Rantibody, preferably an antibody according to the invention, for thetreatment of a patient suffering from rheumatoid arthritis, said patientdo respond moderate or do not respond to the treatment with a TNFantagonist, anti-CD20 antibody, CTLA4Ig or anti-IL6 antibody. A furtherembodiment of the invention is the use of an anti-IL33R antibody,preferably an antibody according to the invention, for the manufactureof a medicament for the treatment of a patient suffering from rheumatoidarthritis, ulcerative colitis or asthma.

The invention further provides a method for the manufacture of apharmaceutical composition comprising an effective amount of an antibodyaccording to the invention together with a pharmaceutically acceptablecarrier and the use of the antibody according to the invention for sucha method. The invention further provides the use of an antibodyaccording to the invention in an effective amount for the manufacture ofa pharmaceutical agent, preferably together with a pharmaceuticallyacceptable carrier, for the treatment of a patient suffering fromrheumatoid arthritis, ulcerative colitis or asthma. The invention alsoprovides the use of an antibody according to the invention in aneffective amount for the manufacture of a pharmaceutical agent,preferably together with a pharmaceutically acceptable carrier, for thetreatment of a patient suffering from rheumatoid arthritis, ulcerativecolitis or asthma.

Description of the Sequences

-   SEQ ID NO: 1 heavy chain CDR3, Mab ra170-   SEQ ID NO: 2 heavy chain CDR2, Mab ra170-   SEQ ID NO: 3 heavy chain CDR1, Mab ra170-   SEQ ID NO: 4 light chain CDR3, Mab ra170-   SEQ ID NO: 5 light chain CDR2, Mab ra170-   SEQ ID NO: 6 light chain CDR1, Mab ra170-   SEQ ID NO: 7 heavy chain variable domain, Mab ra170-   SEQ ID NO: 8 light chain variable domain, Mab ra170-   SEQ ID NO: 9 human γ1 heavy chain constant region with mutations    L234A and L235A (Caucasian Allotype)-   SEQ ID NO: 10 human γ1 heavy chain constant region (Caucasian    Allotype)-   SEQ ID NO: 11 human γ1 heavy chain constant region (Afroamerican    Allotype)-   SEQ ID NO: 12 human κ light chain constant region-   SEQ ID NO: 13 Replaced ST2 sequence fragment 1 for mut1-   SEQ ID NO: 14 Replaced ST2 sequence fragment 2 for mut1-   SEQ ID NO: 15 Replaced ST2 sequence fragment 3 for mut1-   SEQ ID NO: 16 Replaced ST2 sequence fragment 4 for mut1-   SEQ ID NO: 17 IL-1R fragment 1 mut1, replacing ST2 fragment SEQ13-   SEQ ID NO: 18 IL-1R fragment 2 mut2, replacing ST2 fragment SEQ14-   SEQ ID NO: 19 IL-1R fragment 3 mut3, replacing ST2 fragment SEQ15-   SEQ ID NO: 20 IL-1R fragment 4 mut4, replacing ST2 fragment SEQ16-   SEQ ID NO: 21 heavy chain variable domain, humanized ra170    11.12(VH11)-   SEQ ID NO: 22 heavy chain CDR1, Mab ra170 11.12-   SEQ ID NO: 23 heavy chain CDR2, Mab ra170 11.12-   SEQ ID NO: 24 heavy chain CDR3, Mab ra170 11.12-   SEQ ID NO: 25 heavy chain variable domain, humanized ra170    10.12(VH10)-   SEQ ID NO: 26 heavy chain CDR1, Mab ra170 10.12-   SEQ ID NO: 27 heavy chain CDR2, Mab ra170 10.12-   SEQ ID NO: 28 heavy chain CDR3, Mab ra170 10.12-   SEQ ID NO: 29 human IgG4 SPLE backbone (human γ4 heavy chain    constant region with mutations L235E and S228P)-   SEQ ID NO: 30 light chain variable domain, humanized ra170 (11.12    and 10.12)-   SEQ ID NO: 31 light chain CDR1, Mab ra170 10.12 and 11.12-   SEQ ID NO: 32 light chain CDR2, Mab ra170 10.12 and 11.12-   SEQ ID NO: 33 light chain CDR3, Mab ra170 10.12 and 11.12-   SEQ ID NO: 34 human kappa backbone

EXAMPLES

The present invention is described in further detain in the followingexamples which are not in any way intended to limit the scope of theinvention as claimed. All references cited are herein specificallyincorporated by reference for all that is described therein. Thefollowing examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Immunization

Wildtype New Zealand White (NZW) Rabbits (Oryctolagus cuniculus) fromCharles River Laboratories International, Inc. were used forimmunization. They were housed and maintained according to theInstitutional Animal Care and Use committee guidelines and Associationfor Assessment and Accreditation of Laboratory Animal Care (Germany,Europe).

Purified, NS0-derived, secreted soluble form of human IL33R fused to Fcregion of human IgG was solved in NaCl-Histidin puffer pH 6.1 at aconcentration of 1 mg/ml and mixed (1:1) with complete Freund's adjuvant(CFA) till generation of stabile emulsion. Three rabbits received anintra dermal (i.d.) injection of 2 ml of emulsion followed by a secondintra muscular (i.m.) and third subcutaneous (s.c.) injection each with1 ml in one week interval. The fourth i.m. injection of 1 ml wasperformed two weeks later followed by two further s.c. injections of 1ml in four weeks interval. 10 ml peripheral whole blood samples of eachanimal was collected 4-6 days after third, fourth, fifth and sixthinjection and used for single cell sorting in FACS. Additional 0.5 mlserum of each animal was collected at the same time and used for thecharacterization of IL33R specific antibody response.

Antibody_Response

The antibody response to the immunization was determined by serialdilution of sera using IL33R specific ELISA, in which 96-well MaxiSorpmicrotiter plates were coated with 0.3 μg/ml rhIL33R protein incarbonate buffer for 1 h at 37° C. Thereafter the wells were blockedwith PBS supplemented with 1% Crotein C (Roche Diagnostics GmbH, DE)over night at 4° C. For detection, goat anti-rabbit IgG linked to ahorseradish peroxidase (The Jackson Laboratory) was used at 1:16000dilution. BM Blue POD Substrat, precipitating Tetramethylbenzidine(TMB), ready-to-use solution from Roche Diagnostics GmbH, DE, was usedfor visualization. Reaction was stopped via 1N HCl and measured in TecanInfinite by 450/690 nm.

Description of Antibody Selection

Sterile cell culture 6-well plates were coated with 2 μg/ml IL33Rprotein in carbonate buffer (0.1 M sodium bicarbonate, 34 mMDisodiumhydrogencarbonate, pH 9.55) over night at 4° C. Plates werewashed in sterile PBS three times before use. EDTA containing rabbitwhole blood was diluted twofold with 1×PBS before density centrifugationon lympholyte mammal (Cedarlane Laboratories) which was performed toisolate rabbit PBMC. PBMCs were washed twice before staining withantibodies.

EL-4 B5 Medium

RPMI 1640 supplemented with 10% FCS (Hyclone, Logan, Utah, USA), 2 mMGlutamin, 1% penicillin/streptomycin solution, 2 mM sodium pyruvate, 10mM HEPES and 0.05 mM β-mercaptoethanole.

Depletion of Macrophages/Monocytes

Sterile 6-well plates (cell culture grade) were used to depletemacrophages and monocytes through unspecific adhesion. Each well wasfilled at maximum with 4 ml media and up to 6×10⁶ peripheral bloodmononuclear cells from the immunized rabbit and allowed to bind for 1 hat 37° C. in the incubator. 50% of the cells in the supernatant wereused for the panning step; the remaining 50% of cells were kept on iceuntil the immune fluorescence staining.

Enrichment of B Cells on IL33R Protein

6-well tissue culture plates coated with IL33R protein were seeded withup to 6×10⁶ cells per 4 ml medium and allowed to bind for 1 h at 37° C.in the incubator. After the enrichment step on IL33R proteinnon-adherent cells were removed by carefully washing the wells 1-2 timeswith 1×PBS. The remaining sticky cells were detached by trypsin for 10min at 37° C. in the incubator and then washed twice in media. The cellswere kept on ice until the immune fluorescence staining.

Immune Fluorescent Staining and Flow Cytometry

Anti-rabbit IgG FITC used for single cell sorting was from AbD Serotec(STAR121F, Düsseldorf, Germany). For surface staining, cells from thedepletion and panning step were incubated with the optimally dilutedAnti-rabbit IgG FITC antibody in PBS for 30 min rolling in the cold roomat 4° C. in the dark. Following centrifugation, the supernatants wereremoved by aspiration. The PBMCs were subjected to 2 cycles ofcentrifugation and washing with ice cold PBS. Finally the PBMCs wereresuspended in ice cold PBS and immediately subjected to the FACSanalyses. Propidium iodide in a concentration of 5 μg/ml (BD Pharmingen,San Diego, Calif., USA) was added prior to the FACS analyses todiscriminate between dead and live cells. A Becton Dickinson FACSAria™equipped with a computer and the FACSDiva™ software (BD Biosciences,USA) were used to collect and analyse the data.

B Cell Culture

B cell cultures were prepared by a method similar to that described byZubler, et al., Eur. J. Immunol. 14 (1984) 357-363, Zubler, et al., J.Exp. Med. 160 (1984) 1170-1183. Briefly, single sorted B cells werecultured in 96-well plates with 210 μl/well medium with Pansorbin® Cells(1:20000) (Calbiochem (Merck), Darmstadt, Deutschland), 5% rabbitthymocyte supernatant (charge 20080910, production Irmgard Thorey) andgamma-irradiated EL-4-B5 murine thymoma cells (2×10⁴/well) for 7 days at37° C. in an atmosphere of 5% CO₂ in the incubator. B cell culturesupernatants were removed for screening and the cells harvestedimmediately for variable region gene recovery or frozen at −80° C. in100 μl RLT buffer (Qiagen, Hilden, Germany).

Isolation of Ribonucleic Acid (RNA)

The cells from which the RNA had to be isolated were at first pelletedby centrifugation. The cell pellet was lysed by the addition of 100 μlRLT-buffer with 10 μl/ml beta-mercaptoethanol. The cells wereresuspended by multiple mixing with a pipette and transferred to a multiwell plate. The plate was shortly centrifugated at 200×g and frozen at−20° C. The isolation of the RNA was performed with the NucleoSpin® 96RNA kit (Macherey & Nagel) according to the manufacturer's instructions.

Reverse Transcription Polymerase Chain Reaction

The reverse transcription was carried out with SuperScript IIIFirst-Strand Synthesis SuperMix (Invitrogen) according to themanufacturer's instructions.

Polymerase Chain Reaction

The polymerase chain reaction was carried out with AccuPrime PfxSuperMix (Invitrogen) according to the manufacturer's instructions.Light chain and heavy chain variable regions were amplified in separatereactions. PCR-primers were used with 25 bp overlaps to target antibodyexpression vectors. PCR-products were purified by NucleoSpin® 96 ExtractII kit (Macherey & Nagel).

Sequencing and SLIC Cloning

The PCR products were sequenced to determine the DNA-sequences of thevariable regions of heavy and light chains. The PCR-products were clonedinto expression vectors by the so called SLIC-cloning method, which isdescribed by Haun, R. S., et al., in BioTechniques 13 (1992) pp. 515-518and Li, M. Z., et al., in Nature Methods 4 (2007) pp. 251-256. Theplasmids for the antibody expression were linearized by restrictionenzyme digestion. The linearized plasmids were purified by preparativeagarose electrophoresis and extracted from the gel (Qiaquick GelExtraction Kit/Qiagen). The purified plasmids were added to aPCR-protocol using overlapping primers (bay 25 bp) for the PCR-productto be cloned. Both the vector and insert were treated with T4 DNApolymerase (Roche Applied Sciences) in the absence of dNTPs to generateoverhangs, then vector and insert were incubated with RecA (New EnglandBiolabs) protein and ATP to promote recombination. Products weretransformed into E. coli. Plasmid DNAs for light chain and heavy chainswere isolated and each couples were combined for transienttransfections.

Transient Transfection for Antibody Expression in HEK293 Cells

HEK293 cells (Invitrogen) were grown in F17-media (Gibco) to 1×10e6cells/ml. 2×10e6 HEK293 cells were transfected with 1 μg HC+LC plasmidssuspended in 293-free (Novagen) and OptiMEM® (Gibco). After 7 daysincubation supernatants were harvested and analyzed.

Antibodies according to the invention show high quality based on theinhibition of IL33 induced NFkB activation in human UT-7 cells.Preferably the antibodies according to the invention show an IC50 valueof 0.05 nM or lower, and more preferably of 0.03 nM or lower. Inaddition the antibodies according to the invention show valuableproperties in the assay combination of an eosinophil assay, mast cellassay, basophil assay (KU812) and TH₂ assay. It was found thatantibodies which show an inhibition of IL33 induced NFkB activation inhuman UT-7 cells of an IC50 value of 0.05 nM or lower, and morepreferably of 0.03 nM or lower will have especially useful properties inthe treatment of rheumatoid arthritis, ulcerative colitis and asthma.Further preferred are antibodies showing IC₅₀ values of 5 nM or lower inthe eosinophil assay, mast cell assay, Th2 assay, basophil assay (IL-5).

Example 2 Inhibition of IL33 Binding to ST2 (ELISA)

The test was performed on 384 well MaxiSorp™ microtiter plates(Sigma-Aldrich, Nunc. DE, Cat. No. 464718) at RT. After each incubationstep plates were washed 3 times with PBST (Phosphate Buffered SalineTween®-20). At the beginning, plates were coated with 1 μg/ml goatanti-human IgG Fc fragment (Jackson Imm. Res., US, Cat. No. 109-006-170)for at least 2 hours (h). Thereafter the wells were blocked with PBSsupplemented with 0.1% Tween®-20 and 2% BSA (Roche Diagnostics GmbH, DE)for 1 h. 60 ng/ml of recombinant human ST2/IL-1R4 Fc chimera (R&DSystems, UK, Cat. No. 523-ST) or recombinant cyno ST2 Fc chimera wascaptured for 1 h. Dilutions of purified antibodies or supernatants fromhybridoma/B-cells in PBS with 0.5% BSA and 0.05% Tween®-20 wereincubated with the receptor protein for 1 h. Biotinylated human IL33(PeproTech, US, Cat No. 500-P261) was added for an additional hour tobuild up the complex. IL33 was biotinylated with Sulfo-NHS-LC-Biotin(Thermo Scientific Pierce, US, Cat. No. 21327) according to themanufacturer's protocol and purified using Zeba™ Desalt Spin Column(Thermo Scientific Pierce, US, Cat. No. 89889). Binding of thebiotinylated IL33 to the complex was detected with 1:4000 dilutedstreptavidin HRP (Roche Diagnostics GmbH, DE, Cat. No. 11089153001).After 1 h the plates were washed 6 times with PBST and developed withfreshly prepared BM blue POD substrate solution (BM blue:3,3′-5,5′-Tetramethylbenzidine, Roche Diagnostics GmbH, DE, Cat. No.11484281001) for 12 minutes at RT. Absorbance was measured at 370 nm.The negative control was defined without addition of ST2/IL-1R4 proteinand the positive control was defined with all components but withoutantibody.

Antibody ra170 shows an IC50 value of 0.32 nM for inhibition of bindingto human ST2 and 0.13 nM for cynomolgus ST2.

Example 3 Determination of the Affinity of Anti-hST2 Antibodies to HumanST2 ECD (His-Avitag Monomer)

-   Instrument: BIACORE® T100-   Chip: CM4 (GE Healthcare BR-1005-34)-   Coupling: amine coupling-   Buffer: 10 mM phosphate buffered saline including 0.05% Tween20    (PBST), pH 7.4, 37° C.

For affinity measurements 10 μg/ml goat anti human Fcg antibody (JacksonImm. Res., US, Cat. No. 115-005-098) has been coupled to the chipsurface for capturing the antibodies binding to ST2. Monomeric human ST2ECD with 6His Avitag™ (Avidity, LLC, US) was added in variousconcentrations in solution. Association was measured by injection of ST2ECD for a contact time of 120 sec. (single cycle-kinetic) at 37° C.;dissociation was measured by washing the chip surface with buffer for1800 sec. at 37° C. For calculation of apparent K_(D) and other kineticparameters the Langmuir 1:1 model was used. Results (average of twomeasurements) are shown in Table 1.

TABLE 1 Average of affinity data measured by SPR (BIACORE T100) in 10 mMPBST at 37° C., pH 7.4 Antibody app. K_(D) (M) k_(a) (1/Ms) k_(d) (1/s)t_(1/2) (min) ra170 2.7 × 10⁻¹² 4.6 × 10⁶ 1.3 × 10⁻⁵ 881 IgG4 SPLE ra1701.1 × 10⁻⁰⁹ 4.9 × 10⁶ 5.1 × 10⁻³ 2 10.12 ra170 1.4 × 10⁻¹⁰ 3.1 × 10⁶ 4.3× 10⁻⁴ 27 11.12

Example 4 Inhibition of IL33 Induced NFkB Activation in Human UT-7 Cellsa) Reagents:

-   -   UT-7 cell line (DSMZ # ACC 137)    -   Culture medium: RPMI 1640 (Gibco #10509-24) supplemented with 2        mM L-glutamine (Gibco #25030), 1.0 mM sodium pyruvate (Gibco        #11360-039), 0.1 mM NEAA (Gibco #11140-035), 10% FCS (Gibco        #10509-24), 10 units/mL rhGM-CSF (Roche #11115138)    -   Recombinant Human IL33 (PeproTech #200-33)    -   PathScan® Phospho-NFkB p65 (Ser536) Sandwich ELISA Antibody Pair        (Cell Signaling #7834)    -   PathScan® Sandwich ELISA Lysis Buffer (Cell Signaling #7018)    -   rhTNFalpha (Roche Applied Sci #11371843)

b) Procedure:

UT-7 cells were grown in RPMI 1640 supplemented with 2 mM L-glutamine,1.0 mM sodium pyruvate, 0.1 mM NEAA, 10% FCS and 10 units/mL GM-CSF in a7% CO₂/95% air mixture at 37° C. Cells were passaged when they reached adensity of ˜1×10⁶ cells/ml and diluted to a density of 2×10⁵ cells/ml.Cell were used for the NFkB assay 2d after passaging. To determine theeffective concentration for IL33, UT-7 cells were seeded into a 96-wellpolypropylene cell culture plate (8.0E+05 cells/well in a total volumeof 220 μl growth medium) and were stimulated with various concentrationsof recombinant human IL33 (0.1-10 ng/ml) for 15 min at 37° C. Then,plates were centrifuged, washed with ice cold PBS and centrifuged again.PBS was removed and 60 μl PathScan® Sandwich ELISA Lysis Buffer wereadded per well. Cells were incubated with the lysis buffer for 15 min onice. Lysates were cleared by centrifugation and supernatants werecollected. The lysates were stored at −80° C. until determination ofNFkB activation using the PathScan® Phospho-NF-kB p65 ELISA. The ELISAwas performed according to the instructions of the manufacturer The datademonstrated that stimulation of NFkB activation was optimal at 1 ng/mlIL33. For antibody testing, UT7 cells were seeded into a 96-wellpolypropylene cell culture plate (8.0E+05 cells/well) and were incubatedwith different concentrations of antibodies (0.15 ng/ml-300 ng/ml finalconcentration) in a total volume of 220 μl growth medium. Cells wereincubated with the antibodies for 1 h on ice. Subsequently, cells werestimulated with rhIL33 (1 ng/ml final concentration) for 15 min at 37°C. As control, the maximum NFkB activation of UT7 cells was determinedby incubation of UT7 cells with TNF-alpha (30 ng/ml) for 15 min at 37°C. Lysate preparation and NFkB analysis was performed as describedabove. Results are shown in table 2.

TABLE 2 Inhibition of IL33 induced NFkB activation in human UT-7 cellsAntibody NFkB IC50 [nM] ra170 (rabbit IgG1) 0.025 ra170 10.12 0.28 ra17011.12 0.04 Mab523¹ 1.90 AF523 (PAB, goat IgG1)¹ 0.56 HB12 (mouse IgG1)¹1.43 2A5² 66.22 FB9² 0.95 ¹available from R&D Systems ²available fromMBL International Corporation, Order numbers: D065-3, D066-3, D067-3 andABIN130564

Example 5 Binding Site Definition for Clone Ra170

For the definition of the antibody binding sites, human IL33 ReceptorST2 was cloned and expressed. ST2 is composed of 3 domains D1, D2, D3.Thereof a D1D2 variant was generated and residual binding of ra170 wastested. Ra170 binds to ST2 and the truncated variant D1D2.

Binding to the ST2-variant was measured by Surface Plasmon Resonance(SPR) using a BIAcore® T100 instrument (GE Healthcare) at 25° C. TheBIAcore® system is well established for the study of moleculeinteractions. SPR-technology is based on the measurement of therefractive index close to the surface of a gold coated biosensor chip.Changes in the refractive index indicate mass changes on the surfacecaused by the interaction of immobilized ligand with analyte injected insolution. If molecules bind immobilized ligands on the surface the massincreases, in case of dissociation the mass decreases reflecting thecomplex dissociation. SPR allows a continuous real-time monitoring ofligand/analyte bindings and thus the determination of association rateconstants (ka), dissociation rate constants (kd), and equilibriumconstants (KD). Injecting a single concentration value gives a clearstatement about the binding abilities of the analyte, but gives also arough suggestion about the binding values.

Amine coupling of around 8000 resonance units (RU) of a capturing system(capturing ST2-His specific Penta-His, Qiagen, Cat. nr. 34660) wasperformed on a CM5 chip at pH 5.0 using an amine coupling kit suppliedby the GE Healthcare.

For analysis His-tagged ST2-variant was captured by injecting a 300 nMsolution for 1 min at a flow of 30 μl/min. Then the antibody to betested was injected at a concentration of 100 nM for 2 min at a flow of30 μl/min. The dissociation phase was monitored for up to 1 min andtriggered by switching from the sample solution to running buffer. Thesurface was regenerated by 30 sec. washing with a glycin pH2.0 solutionat a flow rate of 30 μl/min.

Bulk refractive index differences were corrected by subtracting theresponse obtained from a blank-coupled surface. Blank injections arealso subtracted (=double referencing).

ST2 variant is bound by antibodies according to the invention comparableto the ST2 wildtype and it is therefore concluded that binding site islocated on the D1D2 domain.

Example 6 Determination of the Binding of Anti-IL33R Antibody TowardsDifferent ST2-Variants

Binding of an antibody according to the invention to differentST2-variants was measured by Surface Plasmon Resonance (SPR) using aBIAcore® A100 instrument (GE Healthcare) at 37° C. The BIAcore® systemis well established for the study of molecule interactions.SPR-technology is based on the measurement of the refractive index closeto the surface of a gold coated biosensor chip. Changes in therefractive index indicate mass changes on the surface caused by theinteraction of immobilized ligand with analyte injected in solution. Ifmolecules bind immobilized ligands on the surface the mass increases, incase of dissociation the mass decreases reflecting the complexdissociation. SPR allows a continuous real-time monitoring ofligand/analyte bindings and thus the determination of association rateconstants (ka), dissociation rate constants (kd), and equilibriumconstants (KD).

Amine coupling of around 800 resonance units (RU) of a capturing system(capturing mFcγ-specific anti-mouse Fcγ, Jackson Imm. Res., Cat. nr.JIR115-005-071 and rbFcg-specific anti-rabbit Fcg, Jackson Imm. Res.,Cat. nr. JIR111-005-046) was performed on a CM5 chip at pH 5.0 using anamine coupling kit supplied by the GE Healthcare.

For analysis different antibodies were captured by injecting a 10 nM ofrabbit antibodies and around 30 nM of mouse antibodies solution for 2min at a flow of 10 μl/min. Then the His tagged ST2 variants to betested were injected at a dilution series with a maximal concentrationof 150 nM for 2.5 min at a flow of 30 μl/min. The dissociation phase wasmonitored for up to 10 min and triggered by switching from the samplesolution to running buffer. The surface of anti-mouse capture antibodieswas regenerated by 60 sec. washing with a glycin pH 1.5 solutionfollowed by 60 sec. washing with a glycine pH2.0 using instrumentalprotocol. The surface of anti-rabbit capture antibodies was regeneratedby two washing steps a 60 sec. with a glycine pH 1.7 solution.

Bulk refractive index differences were corrected by subtracting theresponse obtained from a blank-coupled surface. Blank injections arealso subtracted (=double referencing).

If a ST2 variant is bound by the investigated antibody comparable to ST2wildtype, the variation does not influence the binding of the antibodyto ST2 and it is therefore concluded that binding site is locatedoutside the mutated ST2 region.

If a ST2 variant is not bound by the investigated antibody comparable toST2 wildtype, the variation influences the binding of the antibody toST2 and it is therefore concluded that binding site is located withinthe mutated ST2 region.

Binding properties for antibodies are shown in Table 3. Ra170 binding isinfluenced by Mut2 and Mut3 indicating that its binding site isoverlapping with the mutated sequence stretches. Mab523 binding isinfluenced by Mu2 and Mut3 but also Mut4 indicating a broader bindingregion.

TABLE 3 Binding of anti-IL33R antibody towards different ST2-variantsBinding Antibody Mut1 Mut2 Mut3 Mut4 Ra170 + − − + MAB523 + − − −

TABLE 4 ST2 Mutants IL-1R sequence Replaced ST2 sequence fragment,replacing ST2 Mutant fragment sequence fragment Mut1 SEQ ID NO: 13 SEQID NO: 17 Mut2 SEQ ID NO: 14 SEQ ID NO: 18 Mut3 SEQ ID NO: 15 SEQ ID NO:19 Mut4 SEQ ID NO: 16 SEQ ID NO: 20

Example 7 NK-Assay

IL33 amplifies both T_(H)1 and T_(H)2 responses by activating differentleukocytes and also NK cells (Smithgall, M. D., et al., Int. Immunol. 20(2008) 1019-1030). In the following assay, the secretion of IFN-γ by NKcells was induced by co-culture of IL12 and IL33 and its inhibitionserved as readout during the characterisation of anti-IL33R antibodies.

After isolation of white blood cells (lymphocytes) from healthy blood,NK cells were purified from PBMC using the negative NK cell isolationkit (Miltenyi, #130-092-657). The average purity was >96%.

Reagents

-   -   Human IL-12 (Sigma, #I2276, final concentration [f.c.]=1 ng/ml)    -   Human IL33 (Peprotech, #200-33, f.c.=10 ng/ml)    -   IFN-γ CBA flex set (BD, #558269)    -   NK-cell medium: RPMI 1640 (PAN, # P04-17500), supplemented with        10% FCS (Invitrogen or PAA), 1% sodium pyruvate (Gibco        Invitrogen, #11360) and L-Glutamine (Gibco Invitrogen,        #25030-024), as well as 0.1% β-Mercaptoethanol (Gibco        Invitrogen, #31350-010).

1×10⁵ NK cells/well were seeded into a 96 well flat bottom plate,optionally pretreated with sample or isotype control antibodies atdifferent concentrations and incubated for one hour at 37° C. NK cellswere then stimulated with 10 ng/ml IL33 and 1 ng/ml IL-12 and incubatedfor 20 hrs. After this, supernatants were harvested, centrifugated andtested for IFN-γ production. For IFN-γ quantification the CBA flex setplatform (BD™, using a FACS Canto II) was used. IC50 value wasdetermined to be 0.27 nM for ra170, 1.3 nM for ra170 10.12, 1.8 nM forra170 11.12, 2.3 nM for PAB AF523 and no inhibition for Mab 523.

Example 8 Eosinophil Viability Assay

To describe the impact of IL33 in prolonging eosinophil survival anassay based on Chow, J. Y., et al., Cellular & Molecular Immunology 7(2010) 26-34, was established using freshly isolated eosinophils. As theviability of human eosinophils depends on addition of IL33, thepre-incubation with anti-human IL33R [ST2] mAbs at differentconcentrations inhibited this effect. Granulocytes were isolated fromwhole blood by Ficoll-Paque™ PLUS gradient centrifugation (GEHealthcare, #17-1440-03). After erythrocyte lysis, the sedimented redcell/granulocyte pellet was taken to purify eosinophils via a negativeeosinophil cell isolation kit (Miltenyi Biotec, Bergisch Gladbach,#130-092-010).

Reagents:

-   -   Human IL33 (20 ng/ml)    -   Cell Titer Glo®, Luminescent Cell Viability Assay (Promega,        #7571)    -   Eosinophil cell medium: RPMI 1640 supplemented with FCS, 1%        sodium pyruvate, L-glutamine, and 0.1% β-Mercaptoethanol.

1×10⁵ eosinophils/well were transferred into a 96 well flat bottom platebefore medium containing sample antibody was added [5 μg/ml finalconcentration] and incubated for one hour at 37° C. Then, IL33 was addedat a final concentration of 20 ng/ml and eosinophils were incubated at37° C. in a humidified incubator. After 40 hrs, eosinophil viability wasdetermined. For ra170 an IC₅₀ value of 1.3 nM was found.

Example 9 Primary Mast Cell Assay

Mast cells are central in the development and maintenance of allergicreactions by amplification of both innate and adaptive immune responses.Mast cells are localized in tissues and not in circulating blood. Thus,to obtain human mast cells from blood, CD34⁺ hematopoietic progenitorcells were differentiated into mast cells in the presence of human stemcell factor (SCF), IL-3 and IL-6 within 5-6 weeks. The followingprotocol is based on the publication by Saito, H., et al., NatureProtocols 1 (2006), 2178-2183.

After isolation of white blood cells (see example 10), CD34+hematopoietic progenitor cells were purified from PBMC using the CD34MicroBead kit (Miltenyi Biotec, #130-046-702). The average yield was1-2×10⁵ total cells per donor; usually 50% CD34⁺ CD117⁺ cells wereobtained.

Reagents and Differentiation Protocol:

-   -   Methocult® (Stem Cell Tech., # H4236)    -   Insulin-transferrin-selenium supplement (Invitrogen, #51300-044)    -   BSA, bovine serum albumin    -   Human SCF    -   Human IL3 and IL6    -   Basic Mast Cell medium (bMC): IMDM (Iscove's Modified Dulbecco's        Medium), supplemented with 0.1% β-Mercaptoethanol and 1%        Penicillin/Streptomycin.

After purification, 1.5×10⁵ purified CD34+ cells were resuspended in bMCmedium, supplemented with IL-3, IL-6 and SCF, BSA, Methocult®, andInsulin-transferrin-selenium supplement and seeded into 10-12 wells of a24 well plate and cultured for 5-6 weeks.

Functional Mast Cell Assay

After the expansion and differentiation phase mast cells were used in afunctional assay analyzing the antagonistic impact of anti-IL33Rantibodies.

Reagents and Medium

-   -   20 ng/ml human IL33    -   Human IL-5    -   Human IL-13    -   Basic Mast Cell medium.

10⁵ mast cells/well were seeded into a flat bottom 96 well plate. First,mast cells were pre-treated with sample or isotype control antibodiesfor one hour at 37° C. For IC₅₀ determination antibodies were used atdifferent concentrations, usually starting with a f.c. of 5 μg/ml anddiluted in 1:3 steps. 20 ng/ml IL33 was then added and cells wereincubated for approximately 40 to 48 hrs at 37° C. before (inhibitionof) T_(H)2 cytokine levels were quantified. After the indicatedincubation time, cell suspensions were transferred into a V-bottom plateand sedimented (400×g, 10 min at RT). The supernatant was then used todetermine cytokine levels (IL-5 and IL-13). For ra170 IC₅₀ values up to0.4 nM (IL-13) and 0.2 (IL-5) were found.

Example 10 Human Th2 Assay

Peripheral blood mononuclear cells (PBMC) were isolated from healthyvolunteers by a Ficoll Hypaque density gradient. After washing the cellswith PBS pH 7.2 with 2 mM EDTA, cells were washed once with PBS pH 7.2with 0.5% BSA and 2 mM EDTA. Naive CD4+ T cells were isolated from PBMCusing CD4+ T cell isolation kit II (Miltenyi Biotec) and magneticseparation. The enriched T cells were washed 3 times using completedRPMI 1640 (supplemented with 10% FCS, 2-mercaptoethanol, L-glutamine,sodium pyruvate, non-essential amino acids, andPenicillin/Streptomycin), resuspended and plated at 0.5×10⁶ cells/ml in6 well flat bottom plates, with 1:1 ratio of Dynabeads T-ActivatorCD3/CD28 (Invitrogen), 10 ng/ml of IL-2, 10 ng/ml of IL-4, 5 μg/ml ofanti-IL-12 (R&D System), and 5 μg/ml of anti-IFNγ (R&D System), andincubated for 4 days at 37° C. Cells were split, then kept in the sameculture condition for another 4 days. Cells were washed with completedRPMI two times and then rested at 1×10⁶ cells/ml in completed RPMI with2 ng/ml of IL2 for 3 days. One day before the assay, High-binding96-wells flat-bottom plates (Costar) were coated with 5 μg/ml of solubleanti-CD3e (BD Bioscience). On the assay day, cells were washed withcomplete RPMI twice, and rest for another 4 hours without IL-2. Plateswere washed with PBS three times. 1×10⁵ cells/well were plated incomplete RPMI, supplemented with 1 μg/ml of anti-CD28 (BD Bioscience).Cells were subsequently treated with serial dilution of anti-ST2 Ab orisotype control Ab (0-10 μg/ml) for 30 min, then restimulated with 10ng/ml of IL33 (Peprotech), in a total volume of 200 μl, then cultured at37° C. under 5% CO₂ for 64 hours. Supernatants were collected forIL-5/IL-13 ELISA (R&D Systems). For ra170 IC50 value was determined tobe 2.77±2.58 nM (Mean±SD, n=6) for IL-5, and 1.10±1.05 (Mean±SD, n=5)for IL-13.

Example 11 Cynomolgus NK Cell Assay

Peripheral blood mononuclear cells (PBMC) were isolated from cynomolgusby a Ficoll Hypaque density gradient. After washing the cells with PBSpH 7.2 with 2 mM EDTA, cells were washed once with PBS pH 7.2 with 0.5%BSA and 2 mM EDTA. NK cells were isolated from PBMC following non-humanprimate CD16 Microbeads positive selection after monocytes depletion bynon-human primate CD56 Microbeads (Miltenyi Biotec) and magneticseparation by AutoMACS™ separator. The enriched NK cells were washedthree times using completed RPMI 1640 (supplemented with 10% FCS,2-mercaptoethanol, L-glutamine, sodium pyruvate, non-essential aminoacids, and Penicillin/Streptomycin), resuspended and plated at 2.5×10⁴cells/well in 96 well round bottom plates in complete RPMI. Cells weresubsequently treated with serial dilution of anti-ST2 Ab or isotypecontrol Ab (0-10 μg/ml) for 30 min, restimulated with 20 ng/ml of humanIL33 plus 10 ng/ml of recombinant cynomolgus IL-12 in a total volume of200 μl, then cultured at 37° C. under 5% CO₂ for 24 hours. Supernatantswere collected for cynomolgus IFNγ ELISA (MabTech). IC50 value wasdetermined to be 0.109±0.073 nM (Mean±SD, n=3) for ra170.

Example 12 Basophil Cell Line (KU812) Assay

The human basophil cell line KU812 (ATCC CRL 2099) was cultured in RPMI1640 medium with 10% FBS and Penicillin/Streptomycin. Cells were splittwice a week with cell density not more than 0.5 million cells/ml.Quality controls were routinely performed by FACS analysis of cellssurface expression of c-kit and FceRI. On the assay day, KU812 cells(0.2 million cells/well) were seeded in 96 well round bottom cultureplates with fresh complete medium. Cells were treated with serialdilution of antibodies or control isotypes for 1 hour at 37° C. Aftertreatment, cells were stimulated with 10 ng/ml IL33 (Peprotech)overnight at 37° C. incubator. Cells were spun down and supernatantswere collected for cytokine analysis. Cytokines (IL-5, IL-13, andGM-CSF) were analyzed following the MSD manufactory procedure. For ra170IC50 values from three independent experiments were determined to be3.49±3.43 nM (Mean±SD, IL-13), 1.48±0.75 nM (Mean±SD, IL-5), and1.31±1.22 nM (Mean±SD, GM-CSF). For ra170 IC90 values were determined tobe 7.51±0.99 nM (Mean±SD, IL-13), 4.60±1.65 nM (Mean±SD, IL-5), and9.10±4.74 nM (Mean±SD, GM-CSF).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. An anti-human IL33R antibody, wherein said antibody has a heavy chainvariable domain comprising a CDR3 region of SEQ ID NO:1, a CDR2 regionof SEQ ID NO:2 and a CDR1 region of SEQ ID NO:3 and a light chainvariable domain comprising a CDR3 region of SEQ ID NO:4, a CDR2 regionof SEQ ID NO:5 and a CDR1 region of SEQ ID NO:6, or a chimeric,humanized or T cell epitope depleting antibody variant thereof.
 2. Theantibody according to claim 1, wherein the heavy chain variable domaincomprises SEQ ID NO:7.
 3. The antibody according to claim 2, wherein theheavy chain variable domain comprises SEQ ID NO:7 and the light chainvariable domain comprises SEQ ID NO:8.
 4. The antibody according toclaim 3, wherein said antibody is a chimeric, humanized or T cellepitope depleting antibody variant.
 5. An anti-human IL33R antibody,wherein said antibody has a heavy chain variable domain comprising aCDR3 region of SEQ ID NO:24, a CDR2 region of SEQ ID NO:23 and a CDR1region of SEQ ID NO:22 and a light chain variable domain comprising aCDR3 region of SEQ ID NO:33, a CDR2 region of SEQ ID NO:32 and a CDR1region of SEQ ID NO:31, or a chimeric, humanized or T cell epitopedepleting antibody variant thereof.
 6. A anti-human IL33R antibody,wherein said antibody has a heavy chain variable domain comprising aCDR3 region of SEQ ID NO:28, a CDR2 region of SEQ ID NO:27 and a CDR1region of SEQ ID NO:26 and a light chain variable domain comprising aCDR3 region of SEQ ID NO:33, a CDR2 region of SEQ ID NO:32 and a CDR1region of SEQ ID NO:31, or a chimeric, humanized or T cell epitopedepleting antibody variant thereof.
 7. An antibody according to claim 5,wherein the heavy chain variable domain comprises SEQ ID NO:21.
 8. Anantibody according to claim 7, wherein the heavy chain variable domaincomprises SEQ ID NO:21 and the light chain variable domain comprises SEQID NO:30.
 9. An antibody according to claim 6, wherein the heavy chainvariable domain comprises SEQ ID NO:25.
 10. An antibody according toclaim 9, wherein the heavy chain variable domain comprises SEQ ID NO:25and the light chain variable domain comprises SEQ ID NO:30.
 11. Ananti-human IL33R antibody comprising a human IgG1 or IgG4 isotypemodified in the hinge region at one or more amino acid position between216-240, and/or in the second inter-domain region at one or more aminoacid position between 327-331 between C_(H)2 and C_(H)3, wherein saidantibody is selected from: a) An anti-human IL33R antibody comprising aheavy chain variable domain comprises a CDR3 region of SEQ ID NO:1, aCDR2 region of SEQ ID NO:2 and a CDR1 region of SEQ ID NO:3 and in thatthe light chain variable domain comprises a CDR3 region of SEQ ID NO:4,a CDR2 region of SEQ ID NO:5 and a CDR1 region of SEQ ID NO:6; or b) Ananti-human IL33R antibody comprising a heavy chain variable domaincomprises SEQ ID NO:7, 21 or 25; or c) An anti-human IL33R antibody,wherein the heavy chain variable domain comprises SEQ ID NO:7 and thelight chain variable domain comprises SEQ ID NO:8; or d) An anti-humanIL33R antibody comprising a heavy chain variable domain comprises a CDR3region of SEQ ID NO:24, a CDR2 region of SEQ ID NO:23 and a CDR1 regionof SEQ ID NO:22 and in that the light chain variable domain comprises aCDR3 region of SEQ ID NO:33, a CDR2 region of SEQ ID NO:32 and a CDR1region of SEQ ID NO:31; or e) An anti-human IL33R antibody comprising aheavy chain variable domain comprises a CDR3 region of SEQ ID NO:28, aCDR2 region of SEQ ID NO:27 and a CDR1 region of SEQ ID NO:26 and inthat the light chain variable domain comprises a CDR3 region of SEQ IDNO:33, a CDR2 region of SEQ ID NO:32 and a CDR1 region of SEQ ID NO:31;or f) An anti-human IL33R antibody, wherein the heavy chain variabledomain comprises SEQ ID NO:21 and the light chain variable domaincomprises SEQ ID NO:30; or h) An anti-human IL33R antibody, wherein theheavy chain variable domain comprises SEQ ID NO:25 and the light chainvariable domain comprises SEQ ID NO:30; or i) An anti-human IL33Rantibody that binds to the same epitope as an antibody comprising aheavy chain variable domain comprising SEQ ID NO:7 and a light chainvariable domain comprising SEQ ID NO:8; or j) A chimeric, humanized or Tcell epitope depleting anti-human IL33R antibody variant of any of a-i.12. An anti-human IL33R antibody according to claim 11, wherein saidmutations comprise alanine instead of leucine at amino acid position 234and alanine instead of leucine at amino acid position
 235. 13. Ananti-human IL33R antibody according to claim 11, wherein said mutationscomprise glutamic acid instead of leucine at amino acid position 235 andproline instead of serine at amino acid position
 228. 14. Apharmaceutical composition comprising an anti-human IL33R antibody,wherein said antibody is selected from: a) An anti-human IL33R antibodycomprising a heavy chain variable domain comprises a CDR3 region of SEQID NO:1, a CDR2 region of SEQ ID NO:2 and a CDR1 region of SEQ ID NO:3and in that the light chain variable domain comprises a CDR3 region ofSEQ ID NO:4, a CDR2 region of SEQ ID NO:5 and a CDR1 region of SEQ IDNO:6; or b) An anti-human IL33R antibody comprising a heavy chainvariable domain comprises SEQ ID NO:7, 21 or 25; or c) An anti-humanIL33R antibody, wherein the heavy chain variable domain comprises SEQ IDNO:7 and the light chain variable domain comprises SEQ ID NO:8; or d) Ananti-human IL33R antibody comprising a heavy chain variable domaincomprises a CDR3 region of SEQ ID NO:24, a CDR2 region of SEQ ID NO:23and a CDR1 region of SEQ ID NO:22 and in that the light chain variabledomain comprises a CDR3 region of SEQ ID NO:33, a CDR2 region of SEQ IDNO:32 and a CDR1 region of SEQ ID NO:31; or e) An anti-human IL33Rantibody comprising a heavy chain variable domain comprises a CDR3region of SEQ ID NO:28, a CDR2 region of SEQ ID NO:27 and a CDR1 regionof SEQ ID NO:26 and in that the light chain variable domain comprises aCDR3 region of SEQ ID NO:33, a CDR2 region of SEQ ID NO:32 and a CDR1region of SEQ ID NO:31; or f) An anti-human IL33R antibody, wherein theheavy chain variable domain comprises SEQ ID NO:21 and the light chainvariable domain comprises SEQ ID NO:30; or h) An anti-human IL33Rantibody, wherein the heavy chain variable domain comprises SEQ ID NO:25and the light chain variable domain comprises SEQ ID NO:30; or i) Ananti-human IL33R antibody that binds to the same epitope as an antibodycomprising a heavy chain variable domain comprising SEQ ID NO:7 and alight chain variable domain comprising SEQ ID NO:8; or j) An anti-humanIL33R antibody according to any of a through i comprising a human IgG1or IgG4 isotype antibody modified in the hinge region at one or moreamino acid position between 216-240, and/or in the second inter-domainregion at one or more amino acid position between 327-331 between C_(H)2and C_(H)3; or k) An anti-human IL33R antibody according to j), whereinsaid modification comprise the mutations alanine instead of leucine atamino acid position 234 and alanine instead of leucine at amino acidposition 235; or l) An anti-human IL33R antibody according to j),wherein said modification comprise the mutations glutamic acid insteadof leucine at amino acid position 235 and proline instead of serine atamino acid position 228; or m) A chimeric, humanized or T cell epitopedepleting anti-human IL33R antibody variant of any of a-l; and apharmaceutically acceptable carrier.
 15. A pharmaceutical compositionaccording to claim 14, wherein the antibody binds to the same IL33Repitope to which the antibody according to claim 3 binds.
 16. A methodof treating rheumatoid arthritis, ulcerative colitis or asthmacomprising administering a therapeutically effective amount of ananti-human IL33R antibody to a patient in need of therapy, wherein saidantibody is selected from: a) An anti-human IL33R antibody comprising aheavy chain variable domain comprises a CDR3 region of SEQ ID NO:1, aCDR2 region of SEQ ID NO:2 and a CDR1 region of SEQ ID NO:3 and in thatthe light chain variable domain comprises a CDR3 region of SEQ ID NO:4,a CDR2 region of SEQ ID NO:5 and a CDR1 region of SEQ ID NO:6; or b) Ananti-human IL33R antibody comprising a heavy chain variable domaincomprises SEQ ID NO:7, 21 or 25; or c) An anti-human IL33R antibody,wherein the heavy chain variable domain comprises SEQ ID NO:7 and thelight chain variable domain comprises SEQ ID NO:8; or d) An anti-humanIL33R antibody comprising a heavy chain variable domain comprises a CDR3region of SEQ ID NO:24, a CDR2 region of SEQ ID NO:23 and a CDR1 regionof SEQ ID NO:22 and in that the light chain variable domain comprises aCDR3 region of SEQ ID NO:33, a CDR2 region of SEQ ID NO:32 and a CDR1region of SEQ ID NO:31; or e) An anti-human IL33R antibody comprising aheavy chain variable domain comprises a CDR3 region of SEQ ID NO:28, aCDR2 region of SEQ ID NO:27 and a CDR1 region of SEQ ID NO:26 and inthat the light chain variable domain comprises a CDR3 region of SEQ IDNO:33, a CDR2 region of SEQ ID NO:32 and a CDR1 region of SEQ ID NO:31;or f) An anti-human IL33R antibody, wherein the heavy chain variabledomain comprises SEQ ID NO:21 and the light chain variable domaincomprises SEQ ID NO:30; or h) An anti-human IL33R antibody, wherein theheavy chain variable domain comprises SEQ ID NO:25 and the light chainvariable domain comprises SEQ ID NO:30; or i) An anti-human IL33Rantibody that binds to the same epitope as an antibody comprising aheavy chain variable domain comprising SEQ ID NO:7 and a light chainvariable domain comprising SEQ ID NO:8; or j) An anti-human IL33Rantibody according to any of a thru i comprising a human IgG1 or IgG4isotype antibody modified in the hinge region at one or more amino acidposition between 216-240, and/or in the second inter-domain region atone or more amino acid position between 327-331 between C_(H)2 andC_(H)3; or k) An anti-human IL33R antibody according to j), wherein saidmodification comprise the mutations alanine instead of leucine at aminoacid position 234 and alanine instead of leucine at amino acid position235; or l) An anti-human IL33R antibody according to j), wherein saidmodification comprise the mutations glutamic acid instead of leucine atamino acid position 235 and proline instead of serine at amino acidposition 228; or m) A chimeric, humanized or T cell epitope depletinganti-human IL33R antibody variant of any of a thru l.
 17. The methodaccording to claim 16, wherein the antibody binds to the same IL33Repitope to which the antibody according to claim 3 binds.
 18. A nucleicacid encoding a heavy chain of an antibody binding to IL33R, whereinsaid antibody comprises a heavy chain CDR3 region of SEQ ID NO:1, aheavy chain CDR2 region of SEQ ID NO:2, a heavy chain CDR1 region of SEQID NO:3, and/or a light chain CDR3 region of SEQ ID NO:4, a light chainCDR2 region of SEQ ID NO:5 and a light chain CDR1 region of SEQ ID NO:6.19. A nucleic acid encoding a heavy chain of an antibody binding toIL33R, wherein said antibody comprises a CDR3 region of SEQ ID NO:24, aCDR2 region of SEQ ID NO:23 and a CDR1 region of SEQ ID NO:22 and/or alight chain variable domain comprises a CDR3 region of SEQ ID NO:33, aCDR2 region of SEQ ID NO:32 and a CDR1 region of SEQ ID NO:31.
 20. Anucleic acid encoding a heavy chain of an antibody binding to IL33R,wherein said antibody comprises a CDR3 region of SEQ ID NO:28, a CDR2region of SEQ ID NO:27 and a CDR1 region of SEQ ID NO:26 and/or a thelight chain variable domain comprises a CDR3 region of SEQ ID NO:33, aCDR2 region of SEQ ID NO:32 and a CDR1 region of SEQ ID NO:31.
 21. Anexpression vector comprising a nucleic acid encoding an anti-human IL33Rantibody or antibody fragment thereof comprising a) An anti-human IL33Rantibody comprising a heavy chain variable domain comprises a CDR3region of SEQ ID NO:1, a CDR2 region of SEQ ID NO:2 and a CDR1 region ofSEQ ID NO:3 and in that the light chain variable domain comprises a CDR3region of SEQ ID NO:4, a CDR2 region of SEQ ID NO:5 and a CDR1 region ofSEQ ID NO:6; or b) An anti-human IL33R antibody comprising a heavy chainvariable domain comprises SEQ ID NO:7, 21 or 25; or c) An anti-humanIL33R antibody, wherein the heavy chain variable domain comprises SEQ IDNO:7 and the light chain variable domain comprises SEQ ID NO:8; or d) Ananti-human IL33R antibody comprising a heavy chain variable domaincomprises a CDR3 region of SEQ ID NO:24, a CDR2 region of SEQ ID NO:23and a CDR1 region of SEQ ID NO:22 and in that the light chain variabledomain comprises a CDR3 region of SEQ ID NO:33, a CDR2 region of SEQ IDNO:32 and a CDR1 region of SEQ ID NO:31; or e) An anti-human IL33Rantibody comprising a heavy chain variable domain comprises a CDR3region of SEQ ID NO:28, a CDR2 region of SEQ ID NO:27 and a CDR1 regionof SEQ ID NO:26 and in that the light chain variable domain comprises aCDR3 region of SEQ ID NO:33, a CDR2 region of SEQ ID NO:32 and a CDR1region of SEQ ID NO:31; or f) An anti-human IL33R antibody, wherein theheavy chain variable domain comprises SEQ ID NO:21 and the light chainvariable domain comprises SEQ ID NO:30; or h) An anti-human IL33Rantibody, wherein the heavy chain variable domain comprises SEQ ID NO:25and the light chain variable domain comprises SEQ ID NO:30; or i) Ananti-human IL33R antibody that binds to the same epitope as an antibodycomprising a heavy chain variable domain comprising SEQ ID NO:7 and alight chain variable domain comprising SEQ ID NO:8; or j) An anti-humanIL33R antibody according to any of a through i comprising a human IgG1or IgG4 isotype antibody modified in the hinge region at one or moreamino acid position between 216-240, and/or in the second inter-domainregion at one or more amino acid position between 327-331 between C_(H)2and C_(H)3; or k) An anti-human IL33R antibody according to j), whereinsaid modification comprise the mutations alanine instead of leucine atamino acid position 234 and alanine instead of leucine at amino acidposition 235; or l) An anti-human IL33R antibody according to j),wherein said modification comprise the mutations glutamic acid insteadof leucine at amino acid position 235 and proline instead of serine atamino acid position 228; or m) A chimeric, humanized or T cell epitopedepleting anti-human IL33R antibody variant of any of a through l. 22.The vector according to claim 21, wherein the vector is capable ofexpressing an anti-human IL33R antibody in a host cell.
 23. A host cellcomprising a vector according to claim
 21. 24. The host cell of claim 23wherein the host cell is a prokaryotic cell.
 25. The host cell of claim23 wherein the host cell is a eukaryotic cell.
 26. A method forproducing a recombinant anti-human IL33R antibody, said methodcomprising culturing a host cell according to claim 23 and recoveringsaid antibody from said cell or the cell culture supernatant.